PROJECT SUMMARY/ABSTRACT The long-term goal of this revised U01 proposal is to conduct advanced development and rigorous validation of an emerging circulating trophoblast (cTB)-based noninvasive prenatal diagnostic (NIPD) technology, capable of i) enriching/counting cTBs from maternal blood, and ii) isolating single cTBs for genome-wide detection of fetal genetic abnormalities during the first trimester of pregnancy. An alternative research plan is also presented to explore the use of the same workflow for isolating and characterizing trophoblasts (TBs) in cervix samples. Among potential circulating fetal nucleated cells (CFNCs) in maternal blood, cTBs are an ideal target considering their (i) short lifespan, which excludes the presence of cTBs from prior pregnancies or miscarriages, (ii) representation of fetal karyotype and genotype, and (iii) expression of a unique collection of biomarkers that can be used for both enrichment and identification. However, isolating pure cTBs has been technically challenging due to their extremely low abundance. Over the past decade, Dr. Tseng?s research team at UCLA has developed nanomaterial-embedded diagnostic platforms (a.k.a., NanoVelcro Chips). To exploit the NIPD utility of NanoVelcro Chips, the team first developed a nanoimprinting fabrication process to prepare the laser capture microdissection (LCM)-compatible nanosubstrates in a cost-efficient and scalable manner. These chips, in conjunction with the use of capture and immunocytochemistry (ICC) agents, exhibit superb cTB capture performance. In parallel, high-resolution microscopy imaging and analysis software has been developed to identify and register individual cTBs on the substrates, enabling highly accurate isolation of single cTBs by LCM. In collaboration with Dr. Pisarska, the joint team demonstrated a workflow starting with blood processing, single cTB isolation, and DNA amplification, all the way through whole genome profiling of cTBs by ArrayCGH and/or next generation sequencing. Our central hypothesis is that >10 cTBs can be harvested from 5-mL of maternal blood (>50 TBs from a cervix sample), collected from a pregnant woman during the first trimester of pregnancy (8-12 weeks of gestational age), and whole genome profiling of these cTBs/TBs can be used for diagnosing fetal genetic abnormalities. Over the 5-year funding period, the proposed research will be implemented via two Specific Aims: i) to develop, optimize and validate the proposed cTB-based NIPD technology, and ii) to conduct initial clinical validation in pregnant women recruited from UCLA and CSMC. The joint team envisions that the successful demonstration of the proposed cTBs-based NIPD technology will introduce a revolutionary NIPD solution with the sensitivity and specificity of the gold standard diagnostic tests without the associated risks to the fetus.